The Laboratory Diagnosis of Melioidosis in a Korean Patient

Yong-Woo Shin; Min-Hee Cho; Jeong-Hoon Chun; Changmu Kim; Hee-Bok Oh; Gi-Eun Rhie; Cheon-Kwon Yoo

doi:10.4167/jbv.2011.41.1.19

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Shin, Cho, Chun, Kim, Oh, Rhie, and Yoo: The Laboratory Diagnosis of Melioidosis in a Korean Patient

Original Article

J Bacteriol Virol 2011;41(1):19-25.

Published online: 18 January 2011

DOI: https://doi.org/10.4167/jbv.2011.41.1.19

The Laboratory Diagnosis of Melioidosis in a Korean Patient

Yong-Woo Shin¹, Min-Hee Cho¹, Jeong-Hoon Chun¹, Changmu Kim², Hee-Bok Oh¹, Gi-Eun Rhie¹, Cheon-Kwon Yoo^1,^∗

¹Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Osong Health Technology Administration Complex, Cheongwon, Korea

²Division of Non-Vascular Plants, National Institute of Biological Resources, Korea Environmental Research Complex, Incheon, Korea

^∗Corresponding author: Cheon-Kwon Yoo. Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Osong Health Technology Administration Complex, Cheongwon-gun, Chungcheongbuk-do, 363-951, Korea. Phone: +82-43-419-8270, Fax: +82-43-419-8309 e-mail: ckyoo@nih.go.kr

^∗∗ This work was supported by a Korean National Institute of Health Grant (2008-N00376-00 for C. Y).

Revised 24 November 201024 December 2010 Accepted 29 December 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Burkholderia pseudomallei is a gram-negative opportunistic intracellular pathogen that causes an acute and fatal septicemic melioidosis in humans. The organism is mainly found in Southeastern Asia and Northern Australia. Recently, we encountered a case of melioidosis in a Korean patient and performed the laboratory diagnosis of melioidosis. As a result, a gram negative bacterium was isolated from a melioidosis patient, and it was identified as B. pseudomallei on DNA sequencing of 16S ribosomal RNA with 99.9% homology and biochemical examination of VITEK gram-negative identification card. Also, DNA from cultured bacteria was tested in multiplex PCR, a 245 bp fragment amplified from the metalloprotease gene and a fragment of variable size ranging from 400~700 bp resulting from amplification of the 10 bp repetitive element for B. pseudomallei were confirmed after electrophoresis. The bacterium was sensitive to ceftazidime, imipenem and meropenem but resistant to ticarcillin. So far, there are no domestic cases of melioidosis in Korea, however, due to the increase in international travelers, the incidence of melioidosis is likely to increase. We report a recent case of melioidosis in a Korean patient.

Keywords: Burkholderia pseudomallei, Melioidosis, Diagnosis, VITEK, Multiplex PCR

REFERENCES

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Figure 1.

Colonial growth morphology of Burkholderia pseudomallei. (A) Colonies on BHI agar after 48 h culture; (B) Colonies on MacConkey agar after 48 h culture; (C) Colonies on Ashdown's medium after 48 h culture.

Figure 2.

Comparison of the 16S rRNA sequences from B. pseudomallei (GenBank accession no. CP000572) and B. pseudomallei isolated from a melioidosis patient. Bold letters show the positions of the 3 nucleotide dissimilarities between B. pseudomallei and B. pseudomallei isolated from a melioidosis patient.

Figure 3.

The phylogenetic tree based on the 16S rRNA gene sequences for B. pseudomallei isolate and other Burkholderia. The phylogenetic tree was generated using Neighbor-joining method based on DNA sequence of partial 16S rDNA gene. Bootstrap values of above 50% from a sample of 1,000 replicate are shown on each branch.

Figure 4.

Multiplex PCR analysis of B. mallei, B. thailandensis and B. pseudomallei isolates. Lane 1. B. mallei; lane 2. B. thailandensis; lane 3. B. pseudomallei; lane 4. Genomic DNA isolated directly from blood; lane 5. B. pseudomallei cultured from blood; lane 6. B. pseudomallei cultured from sputum; lane 7. B. cepacia; lane 8. P. aeruginosa; lane 9. E.coli.

Table 1.

Primers used for multiplex PCR

Primer	Sequences	Detected bacteria and size
SR1	5′-ACCGCGTATGAAGGGATGTC-3′	B. thailandensis
SRT3	5′-AAAGCTGCGCGCTCGGCATC-3′	− 308 bp
SR5	5′-ACGCGCACGCACCTGCTGAAC-3′	− 402 bp
14F5	5′-ACCTGCTGCCGGGCTACGACTTCA-3′	B. pseudomallei, B. thailandensis
14R5	5′-CACCTTGCCGACCCACGTAGATGC-3′	− 245 bp

Table 2.

Antimicrobial susceptibility patterns of B. pseudomallei

Antimicrobial agents	Breakpoint for resistance	MIC (μg/ml)	Resistance
Ticarcillin	>64^b	≥128	R
Ticarcillin/clavulanate	≤16/2^b	≤8	S
Ampicillin/sulbactam	ND	4	ND
Piperacillin	≤16^b	≤4	S
Penicillin/tazobactam	≤16/4^b	≤4	S
Cefotaxime	ND	8	ND
Ceftazidime	≤4^b	≤1	S
Cefepime	>8^b	8	I
Aztreonam	>16a	16	I
Imipenem	≤2^b	≤1	S
Meropenem	≤2^b	1	S
Amikacin	≤8^b	≤2	S
Gentamicin	≤4^b	≤1	S
Tobramycin	≤4^b	≤1	S
Ciprofloxacin	≤1^b	1	I
Levofloxacin	≤1^b	1	I
Minocycline	≤4^a	≤1	S
Trimethoprim/sulfamethoxazole	≤4/76^b	≤20	S

^a Breakpoints defined in the general recommendations of the ‘Comite’ de l'Antibiogramme de la Socie'te' Française de Microbiologie' (CASFM).

^b Breakpoints defined for B. cepacia by the CASFM.

R, Resistant; I, Intermediate; S, Susceptible; ND, no interpretative criteria.

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